A versatile and highly efficient method for scarless genome editing in Escherichia coli and Salmonella enterica

Kim, Juhan; Webb, Anthony; Kershner, Jamie P.; Blaskowski, Stephen; Copley, Shelley D.

doi:10.1186/1472-6750-14-84

Cited by 47 publications

(49 citation statements)

References 25 publications

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“…A nonsense mutation in hisG in the ancestral strain of S . enterica was reversed using a genome editing method developed in our laboratory [ 45 ]. The same method was used to delete argC and change the GAA codon specifying Glu382 to GCA specifying Ala to generate the parental strain for this work.…”

Section: Methodsmentioning

confidence: 99%

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Synonymous mutations make dramatic contributions to fitness when growth is limited by a weak-link enzyme

et al. 2018

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Synonymous mutations do not alter the specified amino acid but may alter the structure or function of an mRNA in ways that impact fitness. There are few examples in the literature, however, in which the effects of synonymous mutations on microbial growth rates have been measured, and even fewer for which the underlying mechanism is understood. We evolved four populations of a strain of Salmonella enterica in which a promiscuous enzyme has been recruited to replace an essential enzyme. A previously identified point mutation increases the enzyme’s ability to catalyze the newly needed reaction (required for arginine biosynthesis) but decreases its ability to catalyze its native reaction (required for proline biosynthesis). The poor performance of this enzyme limits growth rate on glucose. After 260 generations, we identified two synonymous mutations in the first six codons of the gene encoding the weak-link enzyme that increase growth rate by 41 and 67%. We introduced all possible synonymous mutations into the first six codons and found substantial effects on growth rate; one doubles growth rate, and another completely abolishes growth. Computational analyses suggest that these mutations affect either the stability of a stem-loop structure that sequesters the start codon or the accessibility of the region between the Shine-Dalgarno sequence and the start codon. Thus, these mutations would be predicted to affect translational efficiency and thereby indirectly affect mRNA stability because translating ribosomes protect mRNA from degradation. Experimental data support these hypotheses. We conclude that the effects of the synonymous mutations are due to a combination of effects on mRNA stability and translation efficiency that alter levels of the weak-link enzyme. These findings suggest that synonymous mutations can have profound effects on fitness under strong selection and that their importance in evolution may be under-appreciated.

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Section: Methodsmentioning

confidence: 99%

“…Construction of S . enterica strains containing mutations in the proAB* operon using the methods described by Kim et al [ 45 ] is described in the Supporting Material.…”

Section: Methodsmentioning

confidence: 99%

Synonymous mutations make dramatic contributions to fitness when growth is limited by a weak-link enzyme

et al. 2018

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“…Site-directed mutagenesis. A modified version of the method described by Kim et al (31) was used for the construction of the rfbSE and rfbJ crossover chromosomal mutants and the rfc frameshift mutant. For the rfbSE and rfbJ crossover chromosomal mutants, mutation cassettes were synthesized by Integrated DNA Technologies, Inc. (USA).…”

Section: Methodsmentioning

confidence: 99%

The O-Antigen Epitope Governs Susceptibility to Colistin in Salmonella enterica

Ricci

Zhang

Teale

et al. 2020

mBio

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Group D and group B Salmonella enterica serovars differ in their susceptibility to colistin with the former frequently intrinsically resistant (MIC > 2 μg/ml); however, the mechanism has not been described. Here, we show that the O-antigen epitope in group D Salmonella governs the levels of colistin susceptibility. Substitution of the rfbJ gene in a group B Salmonella with the rfbSE genes from a group D Salmonella conferred a decrease in susceptibility to colistin. The presence of dideoxyhexose, abequose, and the deoxymannose, tyvelose, differentiate the Salmonella group B and group D O antigens, respectively. We hypothesize that the subtle difference between abequose and tyvelose hinders the colistin molecule from reaching its target. Whole-genome sequencing also revealed that increased colistin susceptibility in a group D Salmonella veterinary isolate was due to a defect in the O-antigen polymerase protein, Rfc. This study shows that two different mechanisms that influence the presence and composition of O antigens affect colistin susceptibility in Salmonella enterica. IMPORTANCE Some serovars of Salmonella, namely, those belonging to group D, appear to show a degree of intrinsic resistance to colistin. This observed intrinsic colistin resistance is of concern since this last-resort drug might no longer be effective for treating severe human infections with the most common Salmonella serovar, Salmonella enterica serovar Enteritidis. Here, we show that the O-antigen epitope in group D Salmonella governs the levels of colistin susceptibility. Using whole-genome sequencing, we also revealed that increased colistin susceptibility in a group D Salmonella veterinary isolate was due to a defect in the O-antigen polymerase protein, Rfc. In summary, we show that two different mechanisms that influence the presence and composition of O antigens affect colistin susceptibility in Salmonella enterica.

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“…Scarless mutations can be obtained when combining this method with a counter-selection marker [14–17]. Currently, λ-Red recombineering is the method of choice for introducing genetic manipulations in S. enterica and E. coli [18] but it has been difficult to implement in several other bacterial species such as Pseudomonas aeruginosa . Recently, CRISPR-Cas has revolutionized eukaryotic genome editing [19–21], but this strategy is more cumbersome for bacteria with limited recombination activities [22–24].…”

Section: Introductionmentioning

confidence: 99%

Efficient Dual-Negative Selection for Bacterial Genome Editing

Cianfanelli

Cunrath

Bumann

2020

Preprint

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11We describe a versatile method for chromosomal gene editing based on classical consecutive 12 single-crossovers. The method exploits rapid plasmid construction using Gibson assembly, a 13 convenient E. coli donor strain, and efficient dual-negative selection for improved suicide vector 14 resolution. We used this method to generate in frame deletions, insertions and point mutations in 15Salmonella enterica with limited hands-on time. Similar strategies allowed efficient gene editing 16 also in Pseudomonas aeruginosa and multi-drug-resistant (MDR) Escherichia coli clinical isolates. 17 18 [19][20][21], but this strategy is more cumbersome for bacteria with limited recombination activities 34 [22][23][24]. 35Here, we combined several improvements for establishing a time-efficient versatile 36 method for consecutive single cross-overs in multiple bacterial species. We used rapid Gibson 37 assembly of PCR products [25] to generate suicide vectors with dual negative selection with I-38SceI and SacB [26, 27] (Fig 1a), which increased counter-selection efficiency to 100% for nearly 39 all tested deletions, insertions and point mutations. We employed an E. coli donor strain that 40 simplifies donor removal after conjugation and avoids common problems with contaminating 100 mM (Sigma Aldrich C1016-500G) containing 15% of glycerol (AppliChem, A1123,1000). 57 Bacteria were resuspended in 5 ml ice-cold CaCl2 100 mM containing 15% of glycerol and 200 µl 58 aliquots were frozen and stored at -80°C. Super-Optimal broth with Catabolite repression (SOC) 59 (tryptone 20 g/L, yeast extract 5 g/L, NaCl 0.5 g/L, KCl 0.186 g/L, MgSO4 4.8 g/L and glucose 3.6 60 g/L) medium was used after heat shock. Kanamycin (Roth T832.4) at a final concentration of 50 61 µg/ml and gentamicin (Gibco 15750-037) at a final concentration of 15 µg/ml were used to select 62 E. coli transformants. For positive selection, kanamycin (Roth T832.4) at a final concentration of 63 50 µg/ml, gentamicin (Gibco 15750-037) at a final concentration of 30 µg/ml, or potassium tellurite 64 (Sigma P0677) at a final concentration of 10 µg/ml, were used to select for S. typhimurium, P.

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A versatile and highly efficient method for scarless genome editing in Escherichia coli and Salmonella enterica

Cited by 47 publications

References 25 publications

Synonymous mutations make dramatic contributions to fitness when growth is limited by a weak-link enzyme

Synonymous mutations make dramatic contributions to fitness when growth is limited by a weak-link enzyme

The O-Antigen Epitope Governs Susceptibility to Colistin in Salmonella enterica

Efficient Dual-Negative Selection for Bacterial Genome Editing

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